Hollow silica nanospheres and methods of making same

ABSTRACT

The disclosure provide hollow nanospheres and methods of making and using the same. The methods and compositions of the disclosure are useful for drug delivery and gene transfer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application Ser. No. 60/955,678, filed Aug. 14, 2007, and toU.S. Provisional Application Ser. No. 61/034,468, filed Mar. 6, 2008,the disclosure of which are incorporated herein by reference.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH

This invention was made with government support awarded by the NationalInstitutes of Health Nanotumor Grant NIH Grant U54 CA 119335. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The disclosure relates to nanostructures and methods of making and usingthe same. More particularly, the disclosure provides hollow nanospheresuseful for drug delivery, imaging, gene transfer and sensing.

BACKGROUND

Traditional drug delivery methods, such as introducing plasmaconcentrations of drugs by injection, or inhalation and ingestion ofdrugs, can require repeated and relatively greater dosing, withproblematic patient compliance. Chemotherapy, which applies thesemethods for cancer treatments, can adversely affect healthy cellsthereby causing serious side effects. Compared with these methods, acontrolled local release system provides the desired constant drugconcentrations at the target specific areas of the body, lowers systemicdrug levels and reduces the potential for harmful side effects. Manymaterials have been developed for drug delivery systems includingliposomes, biodegraded polymer spheres, metal oxides, and otherinorganic particles. Another advanced technology in the medical field isimaging with X-ray or magnetic contrast reagents.

Since chemotherapeutic agents have a reduced efficacy innon-proliferating cells, immunotherapy represents a valuable treatmentoption because it is able to eliminate tumor cells independent of theirproliferative state. Tumor associated antigens have been identified formany tumors and those can serve as target for the immune system. One ofthe approaches used to induce immune responses against cancer cells areDNA vaccines. Injection of plasmids encoding polypeptides can induceimmune responses against the transgene product, offering a potentialmeans for immunization without requiring production and purification ofcomplex antigens. Dendritic cells (DCs) are required to initiate theimmune response to the transgene antigen(s) encoded by such DNA vaccinesand cytotoxic T lymphocytes (CTLs) play a major role in eliminatingmalignant cells by specifically recognizing antigenic peptides presentedon MHC class I molecules by dendritic cells (DC). DNA vaccines althoughshowing some success, are not very efficient and new approaches areneeded to improve efficacy.

SUMMARY

The disclosure demonstrates that “attachment” of

DNA to hollow silica-NPs will increase DNA uptake by taking advantagethe endocytic capacity of DCs. The data provided herein demonstrate thatDCs readily take up DNA that has been adsorbed to hollow Silica-NPs andexpress the encoded transgene. The disclosure provides amulti-functional nanoparticle vaccine/therapy with good prospects fortreatment and prevention of cancers including melanoma. The compositionsand methods are scalable, single agent-multi-functional therapeutics.The approach is modular and useful for delivery of other tumor antigensto treat other cancers.

The disclosure provides methods of synthesis of monodisperse hollowporous nanoparticles and their application in targeted drug and genedelivery. By examining monodisperse nanoparticles, the influence ofnanoparticle size on cellular uptake and in vivo transport can beexamined, as well as potential imaging applications. A key aspect is thedevelopment of synthetic methods that permit differential chemicalfunctionalization of the inner and outer surfaces of the nanoshells. Thegoals are to attach targeting ligands (e.g. integrins, targetingpeptides, or antibodies) to the outer shell surface. The inner surfaceof the nanoshell will be tailored to have hydrophilic, hydrophobic, andacid base properties that optimize binding of a specific payload (e.g.drug, imaging agent, immune stimulant, quantum dot sensor). These havepotential applications in cancer vaccines and drug therapies. Thenanospheres will also be explored in diagnostic schemes as labeledcarriers of PCR primer DNA in the development of array based analysesfor determining genetic mutations in cancer cells. For surfacefunctionalization of silica and titania nanospheres, as well as forsurface modification of biosensing chips, air and water stable reagentsfor self-assembling monolayers are being prepared.

The disclosure demonstrates the synthesis of hollow porous silica andtitania nanospheres in the about 20-1000 um range (e.g., about 40 nm toabout 500 nm range) and shows their use as gene transfer agents and drugdelivery agents to live cells. The data demonstrate the porosity of thenanoshell walls by heavy element staining and high resolutiontransmission electron microscopy. The cellular distribution of thenanoparticles in vivo was characterized by fluorescent imaging. Thecellular distribution of nanoparticle payloads can be characterized byusing two color labeling of the particle and payload. Quantum dots ormetal nanoparticles contained within silica nanoshells can besynthesized. Chemistry techniques can be used to differentiallyfunctionalize the hollow nanoparticle inner and outer surfaces withhydrophobic and hydrophilic functional groups. In one aspect, theparticles can be loaded and then a defined time release of doxorubicinfrom porous hollow nanoparticles can be achieved. Tumor targetingpeptides can be used and attached to the surface of the nanoparticlesfor targeting in a cancer models. Fluorescently labeled nanospheres canbe prepared as carriers of PCR primer DNA into microbubble reactors. Inaddition phosphonate polyethylene glycol (PPEG) reagents can be used asa coating for silica and titania nanospheres for improved in vivobiocompatibility. The disclosure demonstrates how to generated templatesynthesis of monodisperse hollow porous silica and titania nanoshells of45 nm, 100 nm, 200 nm, and 500 nm diameters and with 3-5 nm thick porouswalls; demonstrated uptake of fluorescently labeled 100 nm silica shellsby live human dendritic cells with no cellular toxicity; demonstrationof quantitative plasmid DNA binding to 100 nm silica spheres with theuse of a cationic surface coating; synthesized surface functionalizedsilica nanospheres and demonstrated coupling of surface amino groups tofluorescent dyes; and synthesized phosphonate polyethylene glycol (PEG)reagents that allow coating self-assembled monolayers on silica andtitania surfaces for nonbioadhesive surfaces on biosensor chips.Processing occurs in aerated aqueous solvents at neutral pH for easy andenvironmentally friendly processing.

The disclosure provides a method to synthesize a hollow silicananosphere comprising: (a) synthesizing a precursor of silica shell byhydrolyzing a silicon-containing compound; (b) depositing the precursorof silica shell on a template particle using polyamino acids underneutral condition to give core-shell spheres; (c) removing thepolystyrene core and polyamino acids by calcination or organic solventto provide a hollow silica sphere. In one aspect, the calcinationcomprises heating the core-shell sphere to 450° C. In another aspect,the template particles comprise commercial amine functionalizedpolystyrene beads. In yet another aspect, the precursor of silica shellis deposited on the surface of an amine or carboxylate functionalizedpolystyrene or latex bead. The size of the template particle can be fromabout 40 nm to 1 um. In one aspect the silicon-containing compound isselected from the group consisting of tetraalkoxysilanes,trialkoxysilanes, dialkoxysilanes and any combination thereof. Inanother aspect the silicon-containing compound is selected from thegroup consisting of tetrapropoxysilane, tetraethoxysilane,tetramethoxysilane and any combination thereof. The silicon-containingcompound can be hydrolyzed in acid solution (e.g., hydrochloric acid,sulfuric acid, nitric acid or any combination thereof). Thesilicon-containing compound can be hydrolyzed in 0.01M hydrochloric acidaqueous solution. In one aspect, the final concentration of thesilicon-containing compound in the acids solution is 0.1-10M. In anotheraspect the final concentration of the silicon-containing compound in theacids solution is 1M. In another aspect the polyamino acids includemonopolymer of amino acids with primary amine groups on the backbone insolid or aqueous solution. In another aspect the polyamino acids are0.1% v/w aqueous solution of poly-L-lysine, poly-L-arginine andpolyornithine. In yet another aspect, the silica shell deposits on thesurface of polystyrene beads comprising a polyamino acid. In yet afurther aspect, the deposit of silica shell on the surface ofpolystyrene is conducted at room temperature. In another aspect, thedeposit of silica shell on the surface of polystyrene is conducted undera condition of pH range from 5.5 to 9.5. In yet another aspect, thedeposit of silica shell on the surface of polystyrene is conducted undera condition of pH 7.4. In another aspect, the deposit of silica shell onthe surface of polystyrene is conducted in phosphate buffer. In afurther aspect, the polystyrene core is removed by heating thecore-shell sphere in air at 400-900° C. for 3-6 hours. In one aspect,the heating temperature is achieved by employing a temperature ramp rateof from about 0.1° C./min to about 10° C./min. The polystyrene core canbe removed by washing the core-shell spheres in organic solventsselected from the group consisting of toluene, dichloromethane,chloroform, tetrahydrofuran, dimethylformamide, and any combinationthereof. In one aspect, the polystyrene core is removed by washing thecore-shell spheres in toluene.

The disclosure also provides a hollow sphere made according a methodsdescribed above. In one aspect, the hollow sphere comprises a surfaceamino group or adsorbed polyamines as carriers of an oligonucleotide orpolynucleotide. In another aspect, the hollow spheres are loaded with abiological agent. The biological agent can be a polynucleotide,oligonucleotide, small molecule agent, a peptide or polypeptide and thelike. In another aspect, the hollow spheres can be formulated with apharmaceutically acceptable carrier. The hollow sphere can befunctionalized to associate the hollow sphere with a target analyte orcell

The disclosure also provides methods of nucleic acid delivery comprisinglinking an oligonucleotide or polynucleotide to the hollow sphere of thedisclosure and contacting a cell or subject with the hollowsphere-linked nucleic acid composition

The disclosure also provides a use of polymer template core shell orhollow silica nanoparticles with surface amine groups or adsorbedpolyamines as carriers of DNA for gene transfection.

The disclosure provides an optimized anti-tumor DNA vaccine usingmultifunctional nanoparticles. The hollow silica-NPs are expected tohave very low toxicity and improved degradation profiles compared tosolid particles. The methods and compositions of the disclosuredemonstrate that these hollow silica-NPs readily form complexes withplasmid DNA and can enhance the uptake and expression of plasmid encodedgenes by human dendritic cells (DCs).

In addition, the disclosure provides a peptide (Hp-91) derived from theendogenous molecule HMGB-1, that acts as a potent stimulus for DCactivation and induction of CTL responses in both mouse and humansystems.

Additional aspects of the invention will be understood from thedescription below, the attached drawings and the appended claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic chemical reaction of one method of the disclosure.Schematic the chemical reaction of one method of the disclosure(1=silicic acid, 2=polystyrene or latex beads, 3=polyamino acid orpolyamine coating to aid deposition of silica shell, 4=silica shell,5=hollow silica sphere). For titania spheres the added polyamino acid orpolyamine coating is not needed and Ti(O-t-Bu)4 is the source titaniafor the solution reaction. Not to scale.

FIG. 2 is a photomicrograph of the core-shell polystyrene/silica spheresgiven by the method of the disclosure. (Left) Scanning electronmicroscope photomicrograph of core-shell silica nanoparticles templateon 200 nm beads; (the scale bar is 200 nm). (Right) Transmissionelectron microscopy photo of hollow silica nanoparticles templated on100 nm beads and calcined/burned to remove the polystyrene template;(the scale bar is 100 nm).

FIG. 3 is a photomicrograph of the hollow silica spheres given by themethod of the disclosure. The silica shell is templated by 100 nm beads.(SEM instrument used)

FIG. 4 is a photomicrograph of a hollow silica sphere given by themethod of the present disclosure. The silica shell is templated by 100nm beads. (TEM instrument used for image)

FIG. 5 is a photomicrograph of the hollow titania spheres prepared bythe method of the disclosure. The titania shell shown is templated by200 nm beads. (SEM instrument used)

FIG. 6 is a photograph of DNA adsorbed to hollow silica spheres. 2 μg or6 μg plasmid DNA were complexed with different amounts (0.48-3.8 mg/ml)of either uncharged (top panel) or charged (bottom panel) silicaspheres. Plasmid only, silica spheres only, as well as the complexeswere resolved on a 1% agarose gel at 100V for 1 h. (Geldoc used forimage).

FIG. 7 is a photograph of DNA adsorbed to hollow silica spheres: testingDNA-silica sphere complex stability. Aliquots from each step of thebuffer exchange procedure were collected and resolved on a 1% agarosegel at 100V for 1 h. U=uncharged silica spheres and C=charged silicasphere-DNA complexes.

FIG. 8 shows GFP-expression in silica sphere-DNA complex transfectedDCs. Immature human DCs were exposed to different dilutions of thesilica sphere-DNA complexes. 48 h later Gfp expression was measured byflow cytometry gated on live cells. The histograms depict the relativefluorescence intensity at the different dilutions of charged silicasphere-DNA complexes added to the DCs. B) Percentage of DCs expressingGfp at 48 h after exposure to different dilutions of charged silicasphere-DNA complexes or uncharged silica sphere-DNA complexes. In FIGS.6-8 uncharged is defined as 100 nm silica spheres with the polystyrenecore removed by solvent extraction and charged spheres are defined asthe 100 nm silica spheres with the polystyrene core removed by solventextraction, but containing surface amino group by treatment with(MeO)₃Si(CH₂)₃NH₂.

FIG. 9 shows a photomicrograph of uptake of Silica nanoparticles bydendritic cells. Immature human DCs were exposed to 200 nm FITC-labeledcore-shell Silica-NPs. (left) 2 h after endocytosis, the cells wereimaged in bright-field using an inverted fluorescence microscope (40×magnification). (right) 6 h after exposure, the nuclei were stained withHoechst (blue) and the cells were imaged using a confocal microscope(60× magnification). The white arrows point to the location in thenucleus where nanoparticles were observed.

FIG. 10A-C shows adsorption of DNA to hollow silica-NPs. 2 μg or 6 μgplasmid DNA were complexed with different amounts (0.48-3.8 mg/ml) ofhollow silica-NPs. The same NP concentrations are used in all of thegels. Plasmid only, nanoparticles only, as well as the complexes wereresolved on a 1% agarose gel at 100V for 1 h. A) Comparison of hollowsolvent extracted unmodified and NH2-modified silica NPs. B) Comparisonof burned hollow silica NPs with different surface modifications:unmodified, aminemodified, and poly-L-Lysine (pLL) modified. C)Comparison of DNA adsorption to different size (120, 80, and 45 nm)unmodified solvent extracted silica-NPs.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a nanoparticle”includes a plurality of such nanoparticle and reference to “the cell”includes reference to one or more cells known to those skilled in theart, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The disclosure provides hollow nanospheres, compositions comprising suchhollow nanospheres and methods of using such hollow nanospheres.

Hollow silica nanospheres are potentially applicable to drug deliveryand imaging. Hollow silica nanospheres have uniform and stable wallstructures with excellent long term stability. Their size can becontrolled by using polymer templates for their formation withwell-defined diameters accessible from emulsion polymerization. Theporosity of the silica shell is convenient for loading and releasing ofdrugs or used to contain a heavy element (e.g. metal nanoparticle) ormagnetic oxides for X-ray or magnetic contrast reagents. The surface ofthe hollow silica shell is easily functionalized by graftingbiofunctional groups that may combine with targeting proteins,antibodies, cells, or tissues.

Since chemotherapeutic agents have a reduced efficacy innon-proliferating cells, immunotherapy represents a valuable treatmentoption because it is able to eliminate tumor cells independent of theirproliferative state. Tumor associate antigens have been identified formany tumors and those can serve as target for the immune system. One ofthe approaches used to induce immune responses against cancer cells areDNA vaccines. Injection of plasmids encoding cancer-related antigens orpolypeptides can induce immune responses against the transgene product,offering a potential method for immunization without requiringproduction and purification of complex antigens. Dendritic cells (DCs)are required to initiate the immune response to the transgene antigen(s)encoded by such DNA vaccines and cytotoxic T lymphocytes (CTLs) play amajor role in eliminating malignant cells by specifically recognizingantigenic peptides presented on MHC class I molecules by dendritic cells(DC). DNA vaccines although showing some success, are not very efficientand new approaches are needed to improve efficacy. Hollow silicananoparticles can serve as a platform to deliver DNA vaccines.

Many methods have been employed to fabricate hollow silica spheres, suchas colloidal templating and layer-by-layer (LbL) self-assemblytechniques. Colloidal particles were used to make core-shell nanospheresof gold, silver, CdS, ZnS and polymer beads; however, the inorganictemplates are difficult to remove from the core-shell spheres. For thosehollow spheres templated with polymers, their size and uniformity dependon the species and density of the surface functional groups, which makessize control difficult. The basis of the LbL technique is theelectrostatic attraction between the charged species deposited. But thismethod involves numerous synthetic steps which make large scaleproduction impractical. The challenge of hollow silica nanoparticletechnology is to find a convenient and inexpensive method to fabricatehollow silica nanospheres with uniform, stable shell walls, and at thesame time this shell should have acceptable porosity and a narrow sizedistribution.

There is no scalable inexpensive method for making uniform sizedistributions of hollow nanoparticles. Current nanoparticles used fordrug delivery and sensing are solid. Hollow nanoparticles offer thepossibility of filling with a payload of drug, imaging agent, or othermaterial. The outer and inner surfaces could also be differentiallyfunctionalized.

During the past decade, there has been intense interest about thefabrication of hollow SiO₂ nanoparticles because of their applicationssuch as drug delivery, ultrasound imaging, catalyst, filters, photonicband gap materials. In reported fabrication protocols, colloidaltemplating and layer-by-layer (LbL) self-assembly technique are mostusually used. Colloidal templates used include gold, silver, CdS, ZnSand polymer beads. Polystyrene (PS) beads are attractive nanoscaletemplates since they are inexpensive and their size is easily varied.Furthermore their surface can be functionalized by chemical and physicaltechniques. Finally they are well-suited to make hollow particles sincethe polystyrene template can easily be removed by calcination ordissolution. Calcination can remove the latex cores and give the hollowSiO₂ nanoparticles. For example, the size and the uniformity of thenanoparticles depend in-part upon the density of the surface functionalgroups which makes the size control difficult. Caruso et al reported thefabrication of hollow SiO₂ nanoparticles through the polymer templatedelectrostatic LbL self-assembly of SiO₂ colloid-polymer multilayers,followed by removal of the templated cores. In this study Caruso appliedpoly (diallyldimethylammonium chloride) (PDADMAC), a linear cationicpolyelectrolyte to form the composite multilayer with 25 nm SiO₂colloid. The size of these particles was generally 500 nm and themajority of shells were broken or collapsed when one SiO₂-PDADMAC layerwas applied.

Poly-L-lysine (PL) is one of the simplest polyamino acids with apH-dependent structure and has been applied in many biomimetic synthesesof ordered silica structure.

In addition to the methods of nucleic acid delivery described herein,the disclosure provides a method of synthesis of hollow silicananospheres with controllable size and porous, stable and uniform walls,which are useful for drug delivery and imaging materials.

For example, the DNA-nanosphere complexes of the disclosure takeadvantage of the physiological function of a type of white blood cell,called dendritic cell (DC). DCs are important for initiation of immuneresponses. DCs do not take up non-complexed DNA, and the adsorbtion ofDNA to different size nanospheres allows for effective uptake of DNA andgene expression. The methods and compositions of the disclosure providefor DNA expression in DCs using nanospheres resulting in minimal celldeath and 3-fold more cells that express the transgene using 6-fold lessDNA than current state of the art methods.

In one embodiment, the disclosure provides a hollow silica sphere madefrom a silicon-containing compound with silicon atoms derived from, forexample, tetraalkoxysilanes, silicic acid, sodium silicate and the like.Tetraalkoxysilanes used in disclosure include, for example,tetrapropoxysilane, tetraethoxysilane and tetramethoxysilane. Thedisclosure can include other tetraalkoxysilanes, trialkoxysilanes ordialkoxysilanes. In one embodiment, the silicon-containing compound ishydrolyzed under acidic condition before it reacts to form a silicashell.

The disclosure further provides a method for synthesis of hollow silicaspheres. Commercial polystyrene or latex beads and their amine orcarboxylate functionalized derivatives can be used in the disclosure astemplates. The polymer core template used in the disclosure can have anarrow size distribution and can be chosen from about 10 nm to about 1μm (typically about 20-40, 40-60 or 80-100 nm, but may be larger). Apolyamino acid (e.g., poly-L-lysine), or any other polyamine, can beused in the disclosure with the core template mixture. Asilicon-containing compound is added to react under conditions thatcause the deposition of a silica gel shell on the polystyrene beads toform a uniform silica layer on the template. The polyamino acids can bewashed away after the reaction. The polystyrene core is then removed bycalcinations or solvent extraction. Both methods of core removal providea hollow silica sphere with a uniform, porous, stable silica shell.

The polystyrene beads and the polystyrene or latex beads with amine orcarboxylate functionalized surfaces, which are used in disclosure, canbe purchased from Polysciences Inc and Invitrogen Co. The size oftemplates can be 10 nm, 20 nm, 30 nm, 45 nm, 80 nm, 100 nm, 200 nm, 500nm, 750 nm or 1000 nm and both smaller and larger sized templates can beused (e.g., from about 10 nm to 2000 nm). These beads are monodispersemicrospheres and are packaged as 2.0-4.0% solids (w/v) aqueoussuspensions. These polystyrene microspheres can also contain surfaceprimary amine groups or surface carboxylate groups. The polymer beadsmay also contain a fluorescent dye or other chemical or particle. Thesesizes typically vary by about 10% from batch to batch of manufacturer.After coating using the methods of the disclosure the size increases by10-15 nm, but solvent washing shrinks them slightly and those that arecalcined shrink more. The larger ones tend to shrink more. This occursdue to partial dehydration, as the shell initially forms as a silica gelcoating and on removal of water dehydration to silica of varying degreesof hydration occurs. After calcining they comprise rigid hollow balls ofporous glass like silica that undergo no further or limited size change.

The disclosure provides for the use of polyamine or polyamino acidtemplates, which gives a high yield of well formed spheres. Thepolyamines used in disclosure are homopolymers of amino acids oraliphatic amines with primary amine groups on the polymer backbone. Suchpolyamino acids are poly-L-lysine, poly-L-arginine, and polyornithine,including solids or their aqueous solution, typically about a 0.1%poly-L-lysine aqueous solution. On type of homopolymer of aliphaticamine is polythyleneimine. The polystyrene beads or latex beadsthemselves can template the deposition of a silica shell, but withoutthe presence of polyamine these core-shell spheres have an irregularsilica shell which collapses during the procedure for removing thecores. The concentration of polyamino acids used in the disclosure iskept at low levels to avoid the formation of solid silica spherestemplated by polyamino acids alone, which occurs at higher polyaminoacid concentrations.

As in the sketch of FIG. 1, the polystyrene or latex beads are mixedwith polyamino acids or polyamine before the hydrolyzedtetraalkoxysilane solution is added. The dispersion of beads and 0.1%w/v polyamino acid aqueous solution are added to a phosphate buffer. Theratio of 0.1% w/v polyamino acids and the 2.75% w/v polystyrene beads isfrom 1:1 to 10:1 v/v and most preferably 4:1. The final concentration ofthe polystyrene beads in the buffer solution is from 1:1000 to 1:10000w/v but typically about 1:666 w/v.

One method of the disclosure is depicted in FIG. 1. As shown in FIG. 1,tetraalkoxysilane is hydrolyzed under acidic conditions to form silicicacid (1). Then (1) is added to a mixture of polystyrene or latex beads(2) and polyamino acid or polyamine (3). By selecting appropriatereaction conditions such as temperature, pH, and reaction time thepolycondensation of silicic acid occurs and a silica gel shell (4) isdeposited on the polystyrene beads. The core-shell spheres arecollected, washed and calcined at high temperature to remove the polymercore to give hollow silica (partially dehydrated silica gel) spheres(5).

One method of making a nanostructure of the disclosure is depicted inFIG. 1. Template particle 2 is used in the methods of the disclosure.The template particles can be, for example, a latex or polystyrene bead.The template particle 2 comprises a silicic acid moiety 1. The templateparticle 2 is then treated to comprise a polyamino acid or polyaminegroup 3. The polyamino acid or polyamine group facilitate silicadeposition. A silica shell 4 is then deposited on the template 2. In oneaspect, the template nanostructure is degraded to provide a hollownanostructure of the invention. In other embodiments, the templatenanostructure remains intact. For titania spheres the added polyaminoacid or polyamine coating is not needed and Ti(O-t-Bu)₄ is the sourcetitania for the solution reaction.

The nanostructures may be used with or without decomposing the templatematerial. Batch fabrication is straightforward. The characteristics ofthe resulting hollow sphere make the nanostructures useful forapplication in molecular medicine and in ultrasensitive Raman,biomolecular, and cellular imaging.

Various polymers may be used as the template nanostructure in thegeneration of a nanostructure of the disclosure. For example,o-polyacrylamide and poly(vinyl chloride), poly(vinyl chloride)carboxylated, polystyrene, polypropylene and poly(vinylchloride-co-vinyl acetate co-vinyl) alcohols, may be used.

The ready availability of monosized polystyrene spheres between 45 and500 nm provide a mass produced template for the high yield synthesis ofmono-dispersed hollow silica-NPs with porous shell walls. The polymerspheres readily adsorb a monolayer of poly-L-lysine and other aminopolymers in aqueous solution, which then serve as a basic catalystcoating for the gelation of silicic acid (Scheme I).

The positively charged poly-L-lysine chains in neutral buffer solutionfacilitate the polycondensation reaction of silicic acid. This rapidlyyields a silica shell, which is similar to the neutral conditions usedfor polyamino acid templating of biosilica in organisms, such asdiatoms. The silica gel forms around the poly-L-lysine in a thin (5-10nm) layer on the outer surface of the polystyrene spheres. Theseparticles can be isolated and partially dehydrated by extraction withanhydrous solvents, such as ethanol, to yield stable core shellparticles. The polymer core can be loaded with fluorescent labels totrack the location of the nanoparticles. Dynamic light scatteringmeasurements confirm that the particles can be resuspended by mildultrasonic agitation to produce aqueous colloidal dispersions withsimilar polydispersities as the original polymer beads. The core shellparticles can be heated in air to 450° C., whereupon the polymer coreand poly-L-lysine framework undergo complete oxidation to leave a hollowporous continguous silica gel nanoshell, which is slightly smaller thanthe polymer template. Dehydration of the silica shell on drying causes aslight shrinkage of the gel layer. The method of synthesis, SEM, and TEMimages for two different sizes of hollow nanoparticles that have beenprepared are shown in FIG. 2. Notice the high degree of reproducibilityof the nanoshells in the bulk sample.

FIG. 2 shows a photomicrograph of the core-shell polystyrene/silicaspheres synthesized by the method related to the disclosure. Thecore-shell spheres in the photomicrograph are templated by 100 nm aminefunctionalized polystyrene beads. After coating with the silica shelland drying in vacuum, the diameter of the core-shell spheres is 126±5nm.

Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy was alsoused to monitor removal of the polystyrene core. The C—H (Ar) and —CH₂—of the polystyrene stretching vibrations occur at 3030-2800 cm⁻¹. Theabsorption bands between 1480 and 1400 cm⁻¹ are from C—H bendingvibrations. These features disappear completely after calcination. Whenusing the dissolution method to remove the APS core, the FTIR spectrumshows that about 25% of the polystyrene remained. The amount ofpoly-L-lysine present in all cases was very low and the N—H stretchescould not be observed before or after removal by calcination or organicsolvents.

In the disclosure sodium phosphates can be used to make buffers withdifferent pH. The concentration of the phosphates in buffer can be about0.1M. The deposit of silica shell on polystyrene beads could be given atthe pH range of from 5.5 to 9.5, but typically about pH 7.4. Otherbuffers can be used to modify the pH during silica shell formation.

After the polystyrene or latex beads and polyamino acid or polyamine aremixed in the phosphate buffer, hydrolyzed tetraalkoxysilane is added tothe mixture to deposit silica shell on the beads. The addition ofhydrolyzed tetraalkoxysilane is completed in one portion. The reactionis conducted on an agitator (e.g., a vortex agitator with the vortexspeed of 3000 rpm, which provides vigorous rapid mixing). The vortexmixing time can be from about 2 minutes to 30 minutes, but is typicallyabout 5 minutes. The core-shell spheres are evident by a cloudiness insolution in very short time. Prolonging the vortex mixing time did notincrease the diameter of the core-shell spheres, which indicates thatthe formation of the silica shell on the polystyrene template occurswithin a few minutes.

The reaction is typically conducted at room temperature. The finalconcentration of hydrolyzed tetraalkoxysilane in the reaction system isfrom about 10⁻³M to 5×10⁻³M and typically about 2×10⁻³M. A usefulconcentration of hydrolyzed tetraalkoxysilane provides a uniform andstable silica shell around the templates with narrow size distributionrange, and in high yield based on the template. Higher concentrations ofhydrolyzed tetraalkoxysilane do not give a thicker silica shell butyield solid silica spheres as byproducts.

The core-shell spheres can be isolated from solution by centrifugation.The white precipitate can be washed by being dispersed in deionizedwater and centrifuged. These procedures are followed by washing thespheres with ethanol. These washing procedures in the disclosure are toremove excess reactant and phosphate buffer and are optional. Aftercollection of the pure core-shell spheres by centrifugation, thepolystyrene core can be removed, although it may not be desirabledepending upon further processing or intended use.

Two methods can be used to remove the polystyrene core are calcinationand dissolution, preferably the method of calcination. To remove thecore by dissolution, the core-shell precipitate is suspended in tolueneand the mixture is stirred 1 hour at room temperature and then collectedby centrifugation. The washing procedure is repeated three more timesand then the hollow spheres are washed twice with ethanol. The firstsolvent used in this step may be extended to dichloromethane,chloroform, ethylene diamine, tetrahydrofuran, or dimethylformamide. Thefinal product of the disclosure is obtained by drying at 60° C. undervacuum for 48 hours. To remove the polystyrene cores by calcinations,the core-shell spheres are dried at 60° C. under vacuum for 48 hours,and then heated in air at 400-900° C. for 3-6 hours, more preferablyheating at 450° C. for 4 hours. Temperature ramp and decline rates arefrom 0.1° C./min to 10° C./min, most preferably 5° C./min.

Hollow NP allow for independent surface conjugation of the exterior andinterior surfaces. The hollow silica-NPs have uniform and stable wallstructures with excellent stability for long term storage. Their sizecan be controlled by using polymer templates for their formation withwell-defined diameters accessible from emulsion polymerization. Theporosity of the silica shell is also convenient for loading of smallmolecules, such as drugs or short peptides in the core. The surface ofthe hollow silica-NPs is easily functionalized by grafting functionalgroups (e.g. amino groups as in Scheme II) that may combine withtargeting proteins, antibodies, cells, or tissues. Hollow silica-NPs canalso be made to contain other smaller nanoparticles, including Q-dots tomonitor their position via fluorescence. Polystyrene beads were coatedwith poly-L-Lysine to template 100 nm core-shell silica-NPs; the surfacewas functionalized with (MeO)₃Si(CH₂)₃NH₂ either before or after thepolymer core was removed by calcination or organic solvents (FIG. 1).For the calcinated particles, the surface coating is introduced at theend. The hollow Silica-NPs were functionalized with3-aminopropyl(trimethoxy)silane as in Scheme II to add the additionalamine groups. These are referred to as amine-modified silica-NPs. Forexample, 1 mg of calcinated hollow silica spheres, prepared from the 100nm templates, was suspended in 2 mL of 1%3-aminopropyl(trimethoxy)silane acetone solution. The mixture wasstirred slowly for 2 hours with a magnetic stirrer followed bycollecting the particles by centrifugation. The collected particles werewashed with ethanol and dried in vacuum for 24 hours at roomtemperature. On a 100 nm sphere about 2400 or more surface attachedamino groups can be obtained. Both small molecules and peptidescontaining a free carboxylic acid moiety can be coupled by the couplingreaction to the surface amines with EDAC(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride) to forman amide bond.

FIG. 3 shows a photomicrograph of the hollow silica spheres given by thedisclosure. The size of the hollow silica spheres is 205±7 nm. FIG. 4 isa photomicrograph of a hollow silica sphere. As FIG. 1 shows, thetetraalkoxysilane is hydrolyzed in aqueous acid solution. The acid usedto hydrolyze the tetraalkoxysilanes is 0.01 M hydrochloric acid aqueoussolution. In the disclosure the acids may be extended to sulfuric acidand nitric acid and by inference any other acid with a noninterferinganion. The tetraalkoxysilane could be tetrapropoxysilane,tetraethoxysilane or tetramethoxysilane, most preferablytetramethoxysilane. In the disclosure the precursor of silica shell maybe extended to trialkoxysilanes or diaalkoxysilanes, and by inferenceany source of silicic acid could be used. The final concentration oftetraalkoxysilane in the acid is about 0.1-10M and but is typicallyabout 1 M. The time of hydrolysis reaction is from about 5 minutes to 60minutes, but typically about 15 minutes. The hydrolysis reaction isconducted at room temperature.

Table 1 shows the variation of the size of hollow silica spheres to thesize of templates and the methods of removing the polystyrene cores.After removing templates the diameter of the silica shell shrinks,depending on conditions for core removal and template size. The hollowsilica spheres made from large templates shrink more than those madefrom small templates. The hollow silica spheres obtained by calcinationshrink more than those prepared by dissolution. The size distributionranges of all of the hollow silica spheres made by the disclosure areless than 10%. Since the initially formed wall consists of silica gel,shrinkage of the wall is expected when the gel dries and partialdehydration occurs during calcinations or extractions with anhydroussolvents.

A final product of the disclosure is a white powder consisting ofnanospheres that is easily suspended in deionized water, neutralphosphate buffer, methanol, ethanol, toluene or dichloromethane.Sonication for 15-30 minutes aids resuspension of the nanoparticles.

In the disclosure the hollow silica spheres obtained are functionalizedwith 3-aminopropyl(trimethoxy)silane. To do the functionalization thehollow silica spheres, after calcinations, are dipped in 1%3-aminopropyl(trimethoxy)silane acetone solution. The ratio of hollowsilica spheres and the 3-aminopropyl(trimethoxy)silane is from 1:10 to1:100, preferably 1:20. The reaction is induced by a magnetic stirrer atroom temperature and the reaction time is from about 30 minutes to 4hours, typically about 2 hours. The amine functionalized hollow spheresare washed by deionized water and ethanol, followed by drying undervacuum at room temperature. The surface functionalization reaction canalso be induced before removing the template with organic solvents. Bythis method the The functionalized reaction occurs predominantly on theoutside surface of the core-shell spheres. After removing the templatethe inside surface of the silica shell can be functionalized withdifferent chemistry. Materials, which could react or interact with thesurface of the hollow silica spheres are used for functionalization and,include trialkoxy- or triaryloxysilanes, dialkoxy- or diaryloxysilanes,alkoxy- or aryloxysilanes, derivatives thereof (i.e., oligametic orpolymeric). For example, 3-mercaptopropyl(triethoxy)silane may reactwith the surface of hollow silica spheres and functionalize the surfacewith thiol groups. Amine groups or thiol groups on the surface of hollowsilica spheres allow coupling with biomaterials, such as antibodies,proteins, enzymes, or DNA. After loading with drugs or heavy gas thesekinds functionalized silica spheres may have diverse applications fortargeted drug delivery or targeted contrast-enhanced imaging. Inaddition, the adsorptive properties of silica gel allow reversibleadsorption of materials, as expected from the properties of silica gellike surfaces. The hollow nature of these nanoparticles also impartsthem with a high surface area, as each particle contains an inner andouter surface.

The polystyrene beads or latex beads with amine functionalized surfacecould also be used as template to prepare hollow TiO₂ spheres using asimilar procedure. The precursor of the TiO₂ shell is titaniumt-butoxide. The synthesis of TiO₂ spheres is conducted in ethanolwithout the addition of polyamine. The ratio of template and titaniumt-butoxide, the temperature and the time of reaction, and the method ofremoving the template are same as with the synthesis of silica spheres.Monodispersed hollow spheres with a uniform and porous TiO₂ wall areobtained with yield of 85-95%. Other metal alkoxides, which undergosol-gel type reactivity are expected to react similarly and form hollowshells either with our without added polyamine template.

Some advantages of the disclosure are: using commercial polystyrenebeads and their amine of carboxylate functionalized derivatives astemplates to prepare silica nanoshells of uniform size with porous wallsin high yield. These hollow nanoparticles could be prepared on a largescale and their size could be controlled from 40 nm to 1 um. The abilityto surface functionalize the silica shell will allow diverseapplications of the hollow silica nanospheres. Dissolution of thepolymer core under mild conditions should allow differentialfunctionalization of the hollow shell inner and outer surfaces.

The disclosure also provides a method to functionalize the surface ofhollow silica spheres. For example, a 3-aminopropyl(trimethoxy)silane isused to react with the SiO₂ shell to provide an amine functionalizedsurface, which can then be crosslinked to proteins or used to adsorb DNAfor diverse biological applications. This functionalization can beinduced before or after removing of the template by organic solvents orafter removing the template by calcination.

In yet another aspect, a metal particle or metal containing material canbe incorporated into the hollow silica nanosphere. In this aspect, anaqueous colloidal suspension of a metal oxide nanoparticle precursor isadded to a polyamino polystyrene composition, prior to contacting with asilicon containing compound.

The disclosure further provides a method for adsorbing DNA to hollowsilica nanospheres. These complexes can be used to deliveroligonucleotide or polynucleotides (e.g., DNA or RNA or analogs thereof)into mammalian cells in a tissue culture dish for transgene expressionas well as for vaccine purposes in vivo. For example, the DNA canencodes genes for cancer vaccines or viral or bacterial vaccines forprevention and therapy.

The disclosure provides nanostructures that are biocompatible and can be“loaded” with biological agents or other materials (e.g., drugs,metallic compositions, magnetic compositions and the like).

Although the specific examples provided herein demonstrate particularaspects of the hollow nanostructure of the disclosure, one of skill inthe art will recognize that the size, shape, and layer thickness can allbe individually controlled. Owing to its hollowness, the inner and outersurfaces can be modified with different materials for a wide variety ofcharacteristics and functions.

The nanostructures of the disclosure are biocompatible, and thus can bebiofunctionalized and applied in real-time biomolecular imaging as wellas drug delivery. The term “functionalized” is meant to includefunctional groups attached to the surface of a nanostructure of thedisclosure.

The nanostructures of the disclosure can optionally be functionalized byimprinting functional groups, such as antibodies, proteins, nucleicacids, and the like. Such nanostructures are particularly useful formolecular diagnostics and drug delivery. For example, to prolong ortarget analyte interaction with the hollow nanoparticle surface, abinding agent/targeting domain can be used to promote interaction of ananostructure with a desired target.

In other embodiments, nanostructures of the disclosure are coated toinhibit the accumulation of biological material (e.g., proteinaceousagents) on the nanostructure's surface. In some embodiments,polyethyleneglycol (PEG) is immobilized on nanostructure surfaces toprevent nonspecific interactions.

Attached functional groups can comprise components for specifically, butreversibly or irreversibly, interacting with the specific analyte (e.g.,can be labeled for site/molecule directed interactions). For example, asurface bound functional group (e.g., a targeting ligand) can beattached to a nanostructure of the disclosure. For example, a chemicalmolecule can be immobilized on the surfaces of a nanostructure of thedisclosure.

A targeting ligand can include a receptor bound to the surface of ananostructure of the disclosure that interacts reversibly orirreversibly with a specific analyte. Examples of functional groups(e.g., targeting ligands) include antigen-antibody pairs,receptor-ligand pairs, and carbohydrates and their binding partners. Thebinding ligand may be nucleic acid, when nucleic acid binding proteinsare the targets. As will be appreciated by those in the art, thecomposition of the binding ligand will depend on the composition of thetarget analyte. Binding ligands to a wide variety of analytes are knownor can be readily identified using known techniques.

For example, when the analyte is a single-stranded nucleic acid, thebinding/targeting ligand is generally a substantially complementarynucleic acid. Similarly the analyte may be a nucleic acid bindingprotein and the capture binding ligand is either a single-stranded ordouble-stranded nucleic acid; alternatively, the binding ligand may be anucleic acid binding protein when the analyte is a single ordouble-stranded nucleic acid. When the analyte is a protein, the bindingligands include proteins or small molecules. For example, when theanalyte is an enzyme, suitable binding ligands include substrates,inhibitors, and other proteins that bind the enzyme, i.e. components ofa multi-enzyme (or protein) complex. As will be appreciated by those inthe art, any two molecules that will associate, may be used, either asthe analyte or the functional group (e.g., targeting/binding ligand).Suitable analyte/binding ligand pairs include, but are not limited to,antibodies/antigens, receptors/ligand, proteins/nucleic acids; nucleicacids/nucleic acids, enzymes/substrates and/or inhibitors, carbohydrates(including glycoproteins and glycolipids)/lectins, carbohydrates andother binding partners, proteins/proteins; and protein/small molecules.In one embodiment, the binding ligands are portions (e.g., theextracellular portions) of cell surface receptors.

The disclosure provides nanostructures that have use in the detection ofanalytes in the environment, including explosive and biological agentsas well as in vivo. Accordingly, the invention is useful in HomelandSecurity and the military for detection of analytes as well as formedical diagnostics. In one embodiment, the disclosure provides kits formonitoring military personnel in a war situation where they may beexposed to toxins. The nanostructures are administered or contacted withthe subject prior to potential exposure. The subjects can then bemonitored at set intervals using a detection device.

Commercial applications include environmental toxicology, materialsquality control, food and agricultural products monitoring, anestheticdetection, automobile oil or radiator fluid monitoring, hazardous spillidentification, medical diagnostics, detection and classification ofbacteria and microorganisms both in vitro and in vivo for biomedicaluses and medical diagnostic uses, infectious disease detection, bodyfluids analysis, drug discovery, telesurgery, illegal substancedetection and identification, and the like.

Applications for the nucleic acid constructs provided herein includeselective treatment of cancer, viral infection, genetic diseases,nucleic acid delivery for research and the like.

Provided herein are hollow nanospheres than can be used for the deliveryof biological agents to a cell in vitro or in vivo. The biological agentcan be a nucleic acid (e.g., a polynucleotide, oligonucleotide, peptideor polypeptide). In one aspect, a nucleic acid of interest is conjugatedor operably linked to a hollow nanosphere of the disclosure.

An isolated nucleic acid construct refers to an oligonucleotide orpolynucleotide associated with a hollow nanopshere of the disclosure.For example, a nucleic acid construct includes, but is not limited to,an oligonucleotide or polynucleotide associated with hollow silicananopshere as described herein either directly or via a functionallinker. An oligonucleotide or polynucleotide in the nucleic acidconstructs of the disclosure include fusion polypeptides or peptides,chemical moieties that reduce the net anionic charge of anoligonucleotide or polynucleotide and combinations thereof.

The term polynucleotide(s) and oligonucleotide(s) generally refers toany polyribonucleotide or polydeoxyribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. Thus, for instance, anoligonucleotide as used herein refers to, among others, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions.Thus, a oligonucleotide can comprise an siRNA, an antisense molecule, aribozyme and the like. For example, in one aspect of the disclosure thesiRNA can comprise (fomivirsen) an antisense drug to treat a conditioncalled cytomegalovirus (CMV) retinitis. Other suitable siRNA moleculeswill be apparent to those of skill in the art. Furthermore, it will berecognized that expression vectors or gene delivery constructionscomprising DNA, RNA, a combination of DNA and RNA, and vectors orconstructs comprising nucleic acid analogs can be adsorbed to the hollowsilica nanospheres of the disclosure. The vector or construct cancomprise any of a large number of therapeutic, diagnostic or researchgenetic sequences encoding enzymes, inhibitors, antisense, siRNA,ribozymes, and therapeutic proteins known in the art, such molecules areknown or easily identified in the art (e.g., GFP, growth factors,enzymes, soluble domains of receptor ligands and the like).

In addition, a polynucleotide or oligonucleotides also includestriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules.

In some aspects a polynucleotide or oligonucleotide includes DNAs orRNAs as described above that contain one or more modified bases. Thus,DNAs or RNAs with backbones comprising unusual bases, such as inosine,or modified bases, such as tritylated bases, are polynucleotides oroligonucleotides as the term is used herein.

As used herein, a nucleic acid domain, used interchangeably witholigonucleotide or polynucleotide domain, can be any oligonucleotide orpolynucleotide (e.g., a ribozyme, antisense molecule, polynucleotide,oligonucleotide and the like). Oligonucleotides or polynucleotidesgenerally contain phosphodiester bonds, although in some cases, nucleicacid analogs are included that may have alternate backbones, comprising,e.g., phosphoramidate, phosphorothioate, phosphorodithioate, orO-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press); and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g. to increase thestability and half-life of such molecules in physiological environments.Mixtures of naturally occurring nucleic acids and analogs areencompassed by the term oligonucleotide and polynucleotide;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs can be made.Furthermore, hybrids of RNN, RNB, DNA, and RNA can be used. dsDNA,ssDNA, dsRNA, siRNA are encompassed by the term oligonucleotide andpolynucleotide.

A polynucleotide refers to a polymeric compound made up of any number ofcovalently bonded nucleotide monomers, including nucleic acid moleculessuch as DNA and RNA molecules, including single- double- andtriple-stranded such molecules, and is expressly intended to embracethat group of polynucleotides commonly referred to as“oligonucleotides”, which are typically distinguished as having arelatively small number (no more than about 30, e.g., about 5-10, 10-20or 20-30) of nucleotide constituents.

As used herein, the term “siRNA” is an abbreviation for “shortinterfering RNA”, also sometimes known as “small interfering RNA” or“silencing RNA”, and refers to a class of about 19-25 nucleotide-longdouble-stranded ribonucleic acid molecules that in eukaryotes areinvolved in the RNA interference (RNAi) pathway that results inpost-transcriptional, sequence-specific gene silencing.

The term “dsRNA” is an abbreviation for “double-stranded RNA” and asused herein refers to a ribonucleic acid molecule having twocomplementary RNA strands and which stands distinct from siRNA in beingat least about 26 nucleotides in length, and more typically is at leastabout 50 to about 100 nucleotides in length.

As described above, the nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid may contain combinations ofdeoxyribo- and ribo-nucleotides, and combinations of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xanthinehypoxanthine, isocytosine, isoguanine, etc. As used herein, the term“nucleoside” includes nucleotides and nucleoside and nucleotide analogs,and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus, e.g. the individual units of a peptide nucleic acid,each containing a base, are referred to herein as a nucleoside.

The nucleic acid domain of a nucleic acid construct described herein isnot limited by any particular sequence. Any number of oligonucleotide orpolynucleotides useful for diagnostics, therapeutics and research can beused in the methods and compositions of the disclosure. Various sourcesof oligonucleotides and polynucleotides are available to one of skill inthe art. For example, fragments of a genome may be isolated and theisolated polynucleotides modified in accordance with the disclosure toreduce the overall net anionic charge using phosphodiester and/orphosphothioate protecting groups or may be used as a source forextension of the oligonucleotide or polynucleotide using, for example,nucleic acid synthesis techniques known in the art.

Delivery of a polynucleotides can be achieved by introducing thepolynucleotide into a cell using a hollow nanosphere of the disclosure.For example, a construct comprising such a polynucleotide can bedelivered into a cell using a colloidal dispersion of hollownanospheres. Alternatively, a polynucleotide construct can beincorporated (i.e., cloned) into an appropriate vector which is thenlinked to the nanosphere. For purposes of expression, the polynucleotideencoding a fusion polypeptide may be inserted into a recombinantexpression vector. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes that allow phenotypicselection of the transformed cells. Vectors suitable for such useinclude, but are not limited to, the T7-based expression vector forexpression in bacteria (Rosenberg et al., Gene, 56:125, 1987), thepMSXND expression vector for expression in mammalian cells (Lee andNathans, J. Biol. Chem., 263:3521, 1988), baculovirus-derived vectorsfor expression in insect cells, cauliflower mosaic virus, CaMV, andtobacco mosaic virus, TMV, for expression in plants.

Depending on the vector utilized, any of a number of suitabletranscription and translation elements (regulatory sequences), includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, and the like may be used in the expressionvector (see, e.g., Bitter et al., Methods in Enzymology, 153:516-544,1987). These elements are well known to one of skill in the art.

The term “operably linked” and “operably associated” are usedinterchangeably herein to broadly refer to a chemical or physicalcoupling of two otherwise distinct domains that each have independentbiological function. For example, operably linked refers to thefunctional linkage between a regulatory sequence and the polynucleotideregulated by the regulatory sequence. In another aspect, operably linkedrefers to the association of a nucleic acid domain and a transductiondomain such that each domain retains its independent biological activityunder appropriate conditions. Operably linked further refers to the linkbetween encoded domains of the fusion polypeptides such that each domainis linked in-frame to give rise to the desired polypeptide sequence.

Nanoparticles can serve as a delivery platform that allows for“attachment” of DC-stimuli and antigen on the same particles, which canthen be used for induction of immune responses. Silica-NPs appear tohave the lowest toxicity compared to other nanomaterials tested.

The nanostructures of the disclosure can be used in vivo and in vitro todetect, deliver, identify, and/or characterize analytes of interest. Thenanostructures can be used to detect analytes in environmental samplesas well as samples derived from living organisms. As used herein, theterm “sample” is used in its broadest sense. For example, a sample cancomprise a specimen or culture obtained from any source, as well asbiological and environmental samples. Biological samples may be obtainedfrom animals (including humans) and encompass fluids, solids, tissues,and gases. Biological samples include blood products, such as plasma,serum and the like. Environmental samples include environmental materialsuch as surface matter, soil, water, crystals and industrial samples.The nanostructures can be used, for example, in bodily fluids in vivo orin vitro. Such bodily fluids include, but are not limited to, blood,serum, lymph, cerebral spinal fluid, aqueous humor, interstitial fluid,and urine.

Introduction of plasmid DNA into cells for gene expression is beingwidely used in biology. Primary cells like dendritic cells are verydifficult to transfect using methods like lipofection that work verywell for cell lines. Electroporation using a specialized machine andreagents has been commercialized (Amaxa biosystems) and it is currently,next to viral vectors, the best method on the market for introduction ofplasmid DNA into dendritic cells. This application uses electric forceto introduce DNA into DCs; however, this method causes a high number ofcell death. And gene expression peaks at 20 h, and at 48 h nearly 30% ofthe cells are dead and gene expression is reduced to 10% of the cells.

Since immature DCs readily take up particles, the methods andcompositions of the disclosure have taken advantage of DCs naturalfunction and utilized nanoparticles coated with DNA as a “Trojan horse”to deliver nucleic acids into DCs. In vivo, DCs need to encounter boththe antigen and a DC stimulus for optimal induction of antitumorCytotoxic T lymphocytes (CTL) responses. DCs that encounter antigenwithout stimulus can induce immune tolerance, since DCs effectively takeup particles. The disclosure generates multi-functional silica-NPs thatcarry antigen-encoding nucleic acids and the DC stimulus to ensure thegeneration of strong anti-tumor immune responses. Coupling of nucleicacids and an immunostimulatory agent or peptide (e.g., Hp-91) onsilica-NPs improves the efficacy of these multifunctional nanoparticles.Such compositions and methods are demonstrated in the non-limitingexamples below which demonstrate in vitro assays for DC uptake,activation, and CTL induction and in vivo using a mouse melanoma model.

To adsorb a oligo- or poly-nucleotide to hollow SiO₂ spheres, thespheres are resuspended in and appropriate media. In one aspect, thesphere are resuspended in PBS and sonicated for 15 min to several hoursat a setting sufficient enough to resuspend the spheres. The resuspendedspheres are then diluted and mixed with the desired nucleic acidmolecules. For example, different amounts of SiO₂ spheres were dilutedin 700 mM NaCl and 100 μl of the dilution was mixed at 10 μl per minutewith 100 μl of a plasmid dilution under constant vortexing. Adsorbednucleic acids can be identified or purified by filtration or separation.For example, adsorbed nucleic acids was resolved on an agarose gel afterformation of the complexes (FIG. 6). Adsorption of the nucleic acids tothe SiO₂ spheres prevents the DNA from running into the gel and insteadit stays in the wells with the SiO₂ spheres, whereas free unboundplasmid will run as two bands. Uncharged (defined as 100 nm silicaspheres with the polystyrene core removed by solvent extraction) andcharged (defined as the 100 nm silica spheres with the polystyrene coreremoved by solvent extraction, but containing surface amino group bytreatment with (MeO)₃Si(CH₂)₃NH₂) SiO₂ spheres were compared. UnchargedSiO₂ spheres were more effective at adsorbing plasmid DNA. For example,at 0.95 mg/ml the uncharged SiO₂ spheres adsorbed all of the 2 μg DNA,whereas twice the amount of charged SiO₂ spheres was required to adsorbthe same amount of DNA.

To evaluate uptake of SiO₂ sphere-DNA complexes by primary humandendritic cells (DCs), the SiO₂ spheres-DNA complexes were sonicatedbriefly for 15 min at high, spun down at 6000 rpm for 10 minutes, andthe pellet was resuspended in culture media. To test whether the plasmidwould disassociate from the SiO₂ spheres, aliquots were taken beforesonication, post sonication, from the supernatant after the spin, andfrom the resuspended pellet (FIG. 7). At all stages the plasmid DNA wasretained in the wells, indicating that neither the sonication nor thespinning caused the plasmid DNA to disassociate from the SiO₂ spheres.The same was true for charged and uncharged SiO₂ spheres.

Since the plasmid DNA —SiO₂ sphere complexes and was maintained duringexchange of high salt to culture media, examination of whether immaturehuman DCs would take up the complexes and express the transgene wasanalyzed. A plasmid that encodes for gfp was used, because it allows foreasy monitoring of GFP expression by flow cytometry. 10⁵ immature DCswere exposed to 25 μl diluted SiO₂ sphere-DNA complexes for 2 h andsubsequently 1 ml of fresh medium was added to the cells. DCs werecollected 48 h after exposure to the complexes. At 1:10 dilution of thecharged SiO₂ sphere-DNA complex (0.075 ug plasmid DNA per 10⁵ cells), astrong shift in mean fluorescence intensity was observed and 45% of theDCs expressed GFP (FIG. 8). The uncharged SiO₂ sphere-DNA complexes atthe same dilution showed approximately 2-fold lower percentage ofGfp-positive DCs, which correlated with the fact that ˜2-fold lessplasmid was adsorbed to the same number of particles charged SiO₂spheres (see FIG. 6). Using the “state of the art” transfection methodfor DCs, the Amaxa system, which is electroporation with a specializedmachine, only 10-15% of the DCs expressed GFP at 48 h when 0.5 μgplasmid DNA were used per 10⁵ cells, which is 6.6-fold more DNA and thepercentage of cells expressing GFP is 3-fold lower. Thus the SiO₂spheres provide a very powerful tool to introduce a transgene into DCs.

A nanostructure of the disclosure can be formulated with apharmaceutically acceptable carrier, although the nanostructure may beadministered alone, as a pharmaceutical composition.

A pharmaceutical composition according to the disclosure can be preparedto include a nanostructure of the disclosure, into a form suitable foradministration to a subject using carriers, excipients, and additives orauxiliaries. Frequently used carriers or auxiliaries include magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars, talc,milk protein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol, and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents, and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 15th ed., Easton: Mack Publishing Co., 1405-1412, 1461-1487(1975), and The National Formulary XIV., 14th ed., Washington: AmericanPharmaceutical Association (1975), the contents of which are herebyincorporated by reference. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman's, The PharmacologicalBasis for Therapeutics (7th ed.).

The pharmaceutical compositions according to the disclosure may beadministered locally or systemically. By “effective dose” is meant thequantity of a nanostructure according to the disclosure to sufficientlyprovide measurable SERS signals. Amounts effective for this use will, ofcourse, depend on the tissue and tissue depth, route of delivery and thelike.

Typically, dosages used in vitro may provide useful guidance in theamounts useful for administration of the pharmaceutical composition, andanimal models may be used to determine effective dosages for specific invivo techniques. Various considerations are described, e.g., in Langer,Science, 249: 1527, (1990); Gilman et al. (eds.) (1990), each of whichis herein incorporated by reference.

As used herein, “administering an effective amount” is intended toinclude methods of giving or applying a pharmaceutical composition ofthe disclosure to a subject that allow the composition to perform itsintended function.

The pharmaceutical composition can be administered in a convenientmanner, such as by injection (e.g., subcutaneous, intravenous, and thelike), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, thepharmaceutical composition can be coated with a material to protect thepharmaceutical composition from the action of enzymes, acids, and othernatural conditions that may inactivate the pharmaceutical composition.The pharmaceutical composition can also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof, and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The composition will typically be sterile andfluid to the extent that easy syringability exists. Typically thecomposition will be stable under the conditions of manufacture andstorage and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyetheyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating, such as lecithin, by the maintenance of the required particlesize, in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, isotonic agents, for example, sugars, polyalcohols, such asmannitol, sorbitol, or sodium chloride are used in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent that delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thepharmaceutical composition in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the pharmaceutical composition into a sterilevehicle that contains a basic dispersion medium and the required otheringredients from those enumerated above.

The pharmaceutical composition can be orally administered, for example,with an inert diluent or an assimilable edible carrier. Thepharmaceutical composition and other ingredients can also be enclosed ina hard or soft-shell gelatin capsule, compressed into tablets, orincorporated directly into the subject's diet. For oral administration,the pharmaceutical composition can be incorporated with excipients andused in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 1% by weight ofactive compound. The percentage of the compositions and preparationscan, of course, be varied and can conveniently be between about 5% toabout 80% of the weight of the unit.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: a binder, such as gum gragacanth, acacia, corn starch, orgelatin; excipients such as dicalcium phosphate; a disintegrating agent,such as corn starch, potato starch, alginic acid, and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin, or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit.

For instance, tablets, pills, or capsules can be coated with shellac,sugar, or both. A syrup or elixir can contain the agent, sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye, andflavoring, such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, thepharmaceutical composition can be incorporated into sustained-releasepreparations and formulations.

Thus, a “pharmaceutically acceptable carrier” is intended to includesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Supplementary active compounds can also beincorporated into the compositions.

The working examples provided below are to illustrate, not limit, thedisclosure. Various parameters of the scientific methods employed inthese examples are described in detail below and provide guidance forpracticing the disclosure in general.

EXAMPLES

The disclosure demonstrates that a plasmid encoding for the greenfluorescent protein (Gfp) can be used as a reporter to measure geneexpression. This is a commonly used reporter-gene and it enables a fastand convenient readout of the number of cells that express the transgeneby flow cytometry.

The data show that plasmid DNA can be absorbed onto hollow Silica-NPs of45, 80, and 120 nm size. Surface modifications that increase the chargeof the Silica -NPs are evaluated on all sizes to determine if the DNAadsorption can be increased. To add additional amine groups, the hollowSilica-NPs are functionalized with 3-aminopropyl (trimethoxy)silane. Forexample 1 mg of calcinated hollow silica spheres, prepared from the 100nm templates, are suspended in 2 mL of 1%3-aminopropyl(trimethoxy)silane acetone solution. The mixture arestirred slowly for 2 hours with a magnetic stirrer followed bycollecting the particles by centrifugation. The collected particles arewashed with ethanol and dried in vacuum for 24 hours at roomtemperature. Subsequently the particles are heated at 450° C. Surfacemodification by adding poly-L-lysine are performed after the calcinationprocess by briefly incubating the particles in a 0.1% poly-L-lysinesolution. The solution phase size distribution of the nanoparticles alsoare routinely monitored by dynamic light scattering (DLS) measurements.This will give information on the dispersal state of the nanoparticlesjust before their use in biological experiments. As shown in the datadifferent amounts of plasmid DNA are adsorbed to titrated amounts of thedifferent NPs to compare the efficacy of complex formation and toidentify the most effective silica-NP for DNA adsorption. The complexesare resolved on a 1% agarose gel.

Gene expression: Once the optimal silica-NP surface modification isdetermined, DNA is adsorbed to different sizes of the NP (45, 80, and120 nm) to determine which size confers the strongest transgeneexpression. The rationale to compare different sizes is that they couldtraffic differently, for example it is possible that more of the smallerNPs translocate to the nucleus. Immature human DCs are incubated withincreasing doses of the DNA-NP complexes as described in data using aplasmid that encodes for gfp. 48 h after exposure to the DNA-NPcomplexes, Gfp expression are analyzed by flow cytometry. Kinetics oftransgene expression as well as cell viability using propidium iodidestaining are determined by flow cytometry.

Peptide conjugation: To identify which surface modification is bestsuited for peptide conjugation, fluorescently labeled peptides (FITC onthe N-terminus) are conjugated to the different aminefunctionalizedsilica-NPs. The free C-terminus of the peptide will form an amide bondwith the amino groups on the silica-NPs using EDAC as the couplingreagent. Only for the purpose of demonstrating peptide binding to thesesurfaces will 1 μm size silica-NPs be used, as this allows forvisualization and quantification of the bound peptide by microscopy.Once the most effective silica-NP modification for DNA and peptidebinding is identified, binding of peptides and DNA to the same 1 μm sizesilica-NPs is assessed. To determine co-localization FITC labeledpeptide and tetramethyl-rhodaminelabeled-plasmid DNA are used andanalyzed by microscopy. Additionally adsorption of DNA to peptideconjugated silica-NPs of different sizes by gel electrophoresis and zetapotential measurements are taken. After demonstrating peptide and DNAbinding to the same particle by microscopy, the same methods are used togenerate smaller size multi-functional NPs (45, 80, and 120 nm) for theexperiments listed below.

Generation of dendritic cells: To generate human DCs, anonymous Buffycoats from healthy donors are purchased from the San Diego Blood bank.DCs are prepared from peripheral blood monocytes by culturing CD14+monocytes in GM-CSF and IL-4 for 5 to 7 days, at which point they areimmature DCs. To generate mouse DCs, bone marrow-derived dendritic cells(BM-DC) are prepared from C57/BL6 mice. Briefly, single bone marrow cellsuspensions are obtained from femurs and tibias, and then depleted oflymphocytes, granulocytes, and Ia+ cells using a mixture of monoclonalantibodies (anti-CD4, anti-CD8, anti-B220/CD45R, and anti-Ia) for 45minutes on ice, followed by incubation with low-toxicity rabbitcomplement for 30 minutes at 37° C. Cells are resuspended at aconcentration of 10⁶ cells/ml in medium supplemented with recombinantmurine GM-CSF (10 ng/ml) and plated at 3 ml/well in 6-well plates.Floating cells are removed on days 3 and 5 of culture by gentlepipetting and fed with fresh medium. On day 7, the non-adherent andslightly adherent cells are collected for the experiments. Furtherpurification are achieved by positively selecting CD11c+ cells usingmagnetic beads.

Testing activity of peptide-NP complexes: After demonstrating Hp-91peptide binding to the NPs by microscopy, the DC-stimulatory function ofthe NP-bound peptides are evaluated on both mouse and human DCs. Duringthe maturation process DCs up-regulate co-stimulatory and adhesionmolecules and secrete large amounts of inflammatory cytokines.Therefore, DC maturation can easily be measured by flow cytometrylooking at surface molecule expression levels by ELISA measuring theamount of secreted cytokines.

The immature human and mouse DCs are exposed to increasing doses ofsilica-NP-Hp-91 complex, non-complexed silica-NP (negative control),free Hp-91 peptide, or LPS as positive controls. Cell culturesupernatants are analyzed for the presence of inflammatory cytokines byELISA and changes in cell surface molecule expression are monitored byflow cytometry. The same assays are used to determine the extent towhich CD40L plasmid DNA adsorbed to hollow silica-NPs confers DCmaturation.

Intracellular trafficking of the silica-NPs: If different sizesilica-NPs confer different potentials for gene expression,intracellular trafficking of the silica-NPs are analyze to determine apossible mechanism. DCs are exposed to FITC-labeled NPs of differentsizes and harvested at different time points (30 min, 1 h, 2 h, 6, and24 h) after exposure to the NPs. Cells are spun onto glass slides andstained with Hoechst to visualize the nucleus and determine thelocalization of particles in the nucleus. Cells are stained withlysotracker (Molecular probes), which labels endosomes and lysosomes aswell as with an anti-LAMP1 antibody, which reacts with a lysosomalprotein. These experiments shed light on whether particles of certainsizes or modifications get stuck in lysosomes or whether theytranslocate to the nucleus. Furthermore, to determine whether the NPsand DNA traffic together within the cell or whether in certainintracellular milieus the DNA is released from the NPs,tetramethyl-rhodamine-labeled-plasmid DNA are adsorbed to FITC-labeledNPs and intracellular trafficking kinetics are monitored by microscopy.

A higher density of positive charge on the NP surface is expected tocause higher amounts of DNA adsorption. Thus, the NPs with extra aminegroups or with added poly-L-lysine adsorbed on the surface are expectedto bind more DNA. If both the DC-stimulatory peptide and the CD40Lplasmid confer similar DCs maturation capacity.

2 plasmids can also be adsorbed to the NPs, one encoding the melanomaantigen and the other encoding CD40L as the DC stimulatory molecule.Alternatively non-methylated CpG oligonucleotides, which are also strongstimuli for DC activation, are adsorbed to the NPs.

A MART1(=melanoma antigen)-expressing plasmid and a human MART1-specificCD8+ T cell clone are used to evaluate the induction of antimelanoma CTLresponses. The size and surface modification of Silica-NPs that enabledthe most potent gene expression for human DCs is used to adsorb aMART1-encoding plasmid and to covalently attach the DC-stimulatorypeptide Hp-91. Immature human DCs are exposed to increasing doses ofSilica-NP-complexes or non-complexed NPs as control and subsequentlycocultured with a CD8+ MART1-specific T cell clone. T cell activationwill be measured by IFN-γ and granzymeB ELISPOT as well as in CTLassays.

Using the B16-OVA melanoma tumor model, multi-functional silica-NPscarrying the OVA plasmid (as surrogate tumor antigen) and Hp-91 areinjected into mice before injection of B16-OVA tumor cells to measurethe prophylactic effect. To measure their therapeutic potential, thecomplexes will be injected into tumor bearing mice. In both settings theimmunized mice are monitored for the development of anti-OVA immuneresponses and for tumor growth.

Human monocyte-derived DCs, for example, were generated from PBMCsisolated from healthy donors by culturing adherent cells in GM-CSF andIL-4 for 5 to 7 days. SiO₂ nanoparticles were generated using polymerbeads as template and the surface was functionalized with amino silaneor amino phosphoric acid and the polymer core was removed by calcinationor organic solvents. Several nanoparticles were synthesized: withnon-modified surfaces, functionalized with amine groups, orfunctionalized with poly-L-lysine (pLL). DNA adsorption to the NPs wasdetermined by gel eletrophoresis. DNA adsorption to the NPs caused theDNA to be retained in the well, whereas unbound DNA migrated into thegel. NP uptake by DCs: DCs were incubated with NPs for 2 h in a 50 ul at37° C. Subsequently fresh media was added to the cells and geneexpression was analyzed 48 h later. Using these methods the dataindicated that silica-NPs can adsorb plasmid DNA. Surface modificationsincluding amine groups and poly-L-lysine (pLL) that introducedadditional positive charges were tested and lead to increased DNAadsorption to the NPs. Furthermore, DNA could be adsorbed to silica-NPsof different sizes (45, 80, and 120 nm).

To test uptake, immature DCs were exposed to FITC-labeled NPs of 45,100, and 200 nm diameter. All sizes were effectively taken up by DCs andno obvious preference was observed. Both unmodified and amine-modifiedNPs were taken up by DCs, but pLL-modified NPs were not. Although theNPs were found predominantly in the cytoplasm, confocal microscopyindicated that NPs were also present in the nucleus of the DC. DCsloaded with NPs showed high viability for at least 7 days, showing lowtoxicity compared to other transfection methods. A plasmid that encodesfor gfp was used to demonstrate uptake by cells. The plasmid wasadsorbed to the 120 nm non-modified or amine modified-NPs. Immature DCswere exposed to NP-DNA complexes and analyzed for Gfp expression by flowcytometry. Using DNA complexes with amine modified NPs 45% of the DCsexpressed GFP at 48 h. DNA complexes with un-modified NPs showed 50%reduced Gfp expression, which also correlated with the fact that ˜50%less DNA was adsorbed. In contrast to electroporation methods, no deathof DCs and ˜6-fold increase in the percentage of transfected DCs wasobserved at 48 h.

These experiments demonstrate that SiO₂-nanoparticles of 80 and 120 nmare more efficient than electroporation to introduce a transgene intoDCs in vitro and possibly in vivo.

The data show that human immature DCs take up Silica-NP-DNA complexesand express the transgene at 48 h. A MART1(=melanoma antigen)-expressingplasmid and a MART1-specific CD8+ T cell clone are used to evaluate theinduction of human anti-melanoma CTL responses in vitro.

IFN-γ ELISPOT: Hollow silica-NPs are adsorbed with MART1-encodingplasmid (Silica-NP-MART1=negative control), CD40L encoding plasmid(Silica-NP-CD40L=negative control), both plasmids(Silica-NP-MART1/CD40L=new vaccine), the DC-stimulatory peptide Hp-91(Silica-NP-Hp-91=negative control), and MART1-encoding plasmid plusHp-91 (Silica-NP-MART1/Hp-91=new vaccine). Immature human DC areincubated for 2 hours with the NP-DNA complexes and respective controlslisted above. 48 h and 72 h after NP exposure the DCs are co-culturedwith a CD8+ MART1-specific T cell clone for 24 h. As a positive controlfor T cell activation, the immature DCs are exposed to LPS for 48 h andthen pulsed with MART1 peptide (ELAGIGILTV). T cell activation aremeasured by IFN-γ ELISPOT, which detects and quantified the number ofIFN-γ secreting cells used as indication for T cell activation.

CTL assay: Immature human DC are incubated for 2 hours with the NP-DNAcomplexes and respective controls listed above. 48 h and 72 h after NPexposure the DCs are co-cultured with a CD8+ MART1-specific T cell clonefor 24 h. Subsequently the T cell clone are evaluated for the ability tokill MART expressing target cells in a CTL assay. As target cellsantigen processing-deficient HLA-A2.1+ T2 cells pulsed with the MART1peptide (ELAGIGILTV) or irrelevant 9 mer peptides as a control are used.Prior to peptide pulse, the target cells are labeled with the membranedye PKH26 (red) and subsequently pulsed with 50 μg/ml peptide for 2 h.Cells are transferred (10⁴) to round-bottom 96-well plates and varyingnumbers of CTLs are added. The co-cultures are incubated for 4 h at 37°C. At the end of the culture, the cell viability are determined based onthe mitochondrial trans-membrane potential (ΔΨm) using3,3′-dihexyloxacarbocyanine iodine (DiOC6) by flow cytometry. Live cellsare DiOC6-bright, whereas dying and dead cells are DiOC6-dim.OVA-specific killing of target cells are calculated by gating on the redtarget cells using the following equation: (% of DiOC6dim target cellspulsed with peptide)−(% of DiOC6dim target cells pulsed withoutpeptide). In addition a Granzyme B ELISPOT assay are used to determinethe cytolytic potential of the activated T cells. This assay has showngood correlation with Chromium release assays, but lacks the radioactivehazard.

DCs exposed to Silica-NP-MART1, Silica-NP-CD40L, or Silica-NP-Hp-91 arenot expected to cause T cell activation. DCs exposed to themulti-functional Silica-NP-MART1/CD40L as well as theSilica-NP-MART1/Hp-91 are expected to cause strong T cell activation.

Common mouse melanoma model will be used, where the melanoma cell lineB16-OVA are injected into mice. In this melanoma model, the tumor cellsexpress ovalbumin (OVA), which serves as surrogate tumor antigen,allowing for the measurement of OVA-specific immune responses. Thus aplasmid encoding OVA is used.

The Silica-NP size and composition that confers the strongest T cellactivation are used to generate multi-functional NPs containing plasmidDNA that encodes for chicken ovalbumin (OVA), together with CD40Lplasmid or DC-stimulatory peptides. The multi-functional NPs areevaluated in the in vivo model below in both a prophylactic andtherapeutic setting. Most vaccines using “naked” DNA have shownpromising results when injected i.m. Since the goal is to target DCs,which is more likely to be achieved by i.d. injection, the potency ofthe vaccines is compared when given i.d. or i.m. The potency and type ofimmune responses has been shown to correlate with protection from tumorgrowth and tumor rejection. Therefore, the development of an immuneresponses as well as tumor growth are measured in both the prophylacticand therapeutic setting.

It is generally believed that for tumor rejection strong Th1 immuneresponses characterized by CD8 T cells secreting IFN-γ are required,whereas a Th2 response is thought to play a lesser role. In order togain a better understanding of the tumor rejection mechanism cause bythe multi-functional silica-NP vaccine, further characterization of thetype of immune response is performed. In mouse, the production ofantibody isotypes can be used as a surrogate measurement for the type ofimmune response. IgG2a is recognized as characteristic of a Th1response, whereas the production of IgG1 is characteristic of a Th2response. Therefore, the assessment of the type of immune response aredone by measuring IgG1, IgG2a and IgG2b levels by ELISA. The ratio ofIgG2a/IgG1 antibody titers are used as indicator of Th bias.Furthermore, the secretion of cytokines also defines the type of immuneresponse. A Th2 response is characterized by the secretion of Th2 typecytokines, such as IL-4, IL-5, whereas a Th1 type response ischaracterized by the secretion of IL-2 and IFN-γ.

IL-10 and TGF-β are cytokines that contribute to regulatory T cellresponses, which interfere with tumor rejection. Therefore, thesecretion of these cytokines are measured by ELISA as well.

Prophylaxis: Groups of mice will receive the multi-functional silica NPvaccine first and then receive injections of B16-OVA tumor cells. Halfthe mice from each group are sacrificed to measure immune responses andthe other half are monitored for tumor growth. Groups of mice willreceive i.d. or i.m. injections of PBS, Silica-NP without DNA (neg.control), Silica-NP with irrelevant DNA encoding for a non-mammalianantigen e.g. firefly-luciferase (neg. control), Silica-NP-OVA/CD40L,Silica-NP-OVA-Hp91, Silica-NP-OVA, Silica-NP-CD40L, and Silica-NP-Hp91.Two doses of DNA 10 μg and 50 μg are evaluated either adsorbed to NPs oras “naked” DNA. 10 μg OVA protein in IFA are injected as positivecontrol. 7-14 days after the first injection, mice will receive abooster injection identical to the first injection. 10-14 days after thelast immunization, half of the mice from each group will receive s.c.injections of 1×105 B16-OVA tumor cells and the other half aresacrificed to measure the development of immune responses (see below).The remaining mice are observed bi-weekly for tumor appearance bypalpitation. As primary measurement, the time to tumor appearance in thetreated groups compared to the PBS control is monitored. As secondarymeasurement of tumor size using a set of calipers is used. Tumors of atleast 3 mm are scored. Mice are sacrificed if tumors reach 1.5 cm indiameter or at week 6 after injection of tumor cells, at which point allnon-treated mice will have developed tumors.

Therapeutic: Mice will receive s.c. injections of 5×105 B16-OVA tumorcells. Once the tumor reaches 3 mm in size, mice will receive the samei.m. and i.d. injections of the multi-functional silica NPs and controlsas listed above. In both settings, mice are divided randomly into groups(n=10) and, as above, half the mice from each group are sacrificed 10-14days after the final injection to measure the development of immuneresponses and the other half are monitored for tumor growth. To measurethe development of immune memory, mice that show complete rejection ofthe tumor, are challenged with a second injection of 5×105 B16-OVA cells1 month after the final injection of multifunctional silica NPs.

Measurement of immune response: Groups of mice will receive i.m. or i.d.injections as above. The in vivo induced T cell responses are detectedin vitro using a variety of assays. 1) Proliferation assays areperformed by adding OVA257-264 peptides (to stimulate CD8 T cells) andOVA323-339 (to stimulate CD4 T cells), PBS/no peptide (negative control)or ConA (positive control) to unseparated splenocytes, which contain Tcells and antigen presenting cells, and measuring the uptake of[3H]-thymidine after 4 d. 2) Cytokine secretion: To measure T cellresponses, unseparated splenocytes are set up as described above but thepositive control are phorbal myristate acetate (PMA) and solubleanti-CD3, since ConA is not a very potent stimulus for Th2 cytokines.After 24 h, the culture supernatants are assessed for IL-4, IL-2, IL-5,IL-10, TGF-β and IFN-γ levels by ELISA. Furthermore, IFN-γ ELISPOT areused to measure the number of cytokine secreting T cells. To furtherinvestigate the contribution of CD8+ and CD4+ T cells to the cytokinesecretion, either cell type are depleted from the splenocytes usingspecific antibodies, prior to in vitro culture. 3) CTL assays: To expandOVA-specific T cells from the spleens of the immunized mice, thesplenocytes are cultured in 24-well plates for 6 days at a concentrationof 3×10⁶ cells in 1.0 ml of medium with the addition of recombinantmouse IL-2 (50 units/ml) after 24 h of culture. Furthermore, a standardLDH release assay are performed at day 6 to measure cytolytic activity,by incubating the splenocytes with target cells pulsed with and withoutOVA peptide. The absorbance values from supernatants are recorded at OD490 nm. The percent of specific lysis are calculated as follows:(DExp.−ODSpon.E−ODSpon.T/ODMax.T.−ODSpon.T)×100, where ODExp. is the ODrelated to the experimental LDH relase, ODSpon.E. is the OD related tothe spontaneous release of LDH from the effector cells only, ODSpon.T isthe OD related to the spontaneous LDH release from target cells only,and ODMaxT. is the OD related to the maximum LDH release from targetcells using lysis buffer.

In vivo immune response: To measure the immune responses directly invivo, transfer of 2×10⁶ OTI CD8+ T cells into the mice is performed oneday prior to immunization with the silica-NP vaccines. These OTI CD8+ Tcells are harvested from a T cell receptor transgenic mouse (B6background) which are specific for Kb-SIINFEKL of OVA. Prior toinjection, OT-I cells are labeled with Carboxyfluorescein succinimidylester (CFSE), which consists of a fluorescent molecule that stains theplasma membrane. During each round of cell division, relativefluorescence intensity of the dye is decreased by half, allowing us toexamine cell division in vivo. 3 days after injection of OT-cells, theanimals are tested for proliferation of OT-I cells by flow cytometry.

Toxicity: To monitor for potential pathological effects of the silica-NPvaccines, the spleen, liver and kidney are collected at time ofeuthanasia. The organs are weighed and additional tissue analysis areperformed.

Mice that are immunized with DNA-NP-Hp-91-complexes are protected fromtumor growth and reject the established tumor cells.

The effectiveness of the new silica-NP complexes in inducingmelanoma-specific CTL are determined on a quantitative basis as follows:a). In vitro experiments: The target cells are incubated with decreasingconcentrations of the OVA-derived peptides or corresponding nanoparticlebased vaccines. Values obtained from the LDH-release assays and ELISPOTassays are used to construct dose-response curves and these arecompared. IC50 values from a minimum of 5 independent experiments arecompared for statistical significance using the Student t test. b).

Example 1

Preparation of the solution of hydrolyzed tetramethoxysilane. 14.0 mLtetramethoxysilane is added to 100 mL 0.01 M hydrochloric acid. Themixture is stirred at room temperature for 15 minutes. The solution isto be used as the precursor to deposit silica shells directly.

Example 2

Synthesis of core-shell silica spheres with 100 nm amine polystyrenebeads. 4.0 mL of 2.6% w/v 100 nm sized amine polystyrene beads, 16 mL of0.1% poly-L-lysine solution and 75 mL of 0.1 M phosphate buffer aremixed in a 150 mL pear-shaped flask. 2 mL of hydrolyzedtetramethoxysilane is added and the mixture is stirred vigorously with avortex agitator at a speed of 3000 rpm. The stirring lasts 5 minutes atroom temperature and the mixture is transferred into two 50 mLcentrifuge tubes. A white precipitate is collected by centrifugation.The core-shell particles are suspended in deionzed water and stirredwith the vortex agitator for 5 minutes and then spun down again bycentrifugation. The washing procedure is repeated one more time followedby washing with ethanol. 185 mg Core-shell particles are dried in vacuumat 60° C. for 48 hours.

Example 3

Synthesis of core-shell silica spheres with 200 nm amine polystyrenebeads. The synthesis procedure is similar to example 2, except that the100 nm amine polystyrene beads are replaced by 200 nm amine polystyrenebeads.

Example 4

Synthesis of titania core-shell silica spheres with 200 nm aminepolystyrene beads. 2.0 mL of 2.6% w/v 100 nm sized amine functionalizedpolystyrene beads and 80 mL of absolute ethanol are mixed in a 150 mLpear-shaped flask. 2.0 mL of 1 M titanium t-butoxide/ethanol solution isadded and the mixture is stirred vigorously with a vortex agitator at aspeed of 3000 rpm. The stirring lasts 2 minutes at room temperature andthe mixture is transferred into two 50 mL centrifuge tubes. A whiteprecipitate is collected by centrifugation. The core-shell particles aresuspended in absolute ethanol and stirred with the vortex agitator for 5minutes and then spun down again by centrifugation. The washingprocedure is repeated one more. 120 mg Core-shell polystyrene/titaniaparticles are dried in vacuum at 60° C. for 48 hours.

Example 5

Removing polymer cores by calcination. 10 mg of dry core-shell silicaparticles are placed in a furnace and the temperature is raised at aspeed of 5° C./min to 450° C. The core-shell particles are calcinated inair at 450° C. for 4 hours and the temperature is then cooled at a speedof 5° C./min until it reaches room temperature. 3.2 mg of final productof hollow silica spheres is obtained as a white powder. Yield is nearlyquantitative based on the number of template spheres.

Example 6

Removing polymer cores by dissolution in toluene. 10 mg of driedcore-shell spheres are suspended in 20 mL of toluene and the mixture isstirred with a magnetic stirrer for 1 hour. The solid is collected bycentrifugation. The washing procedure is repeated three more timesfollowed by drying the particles in vacuum at 60° C. for 48 hours. 3.7mg of hollow silica spheres is obtained as white powder. Some residualpolystyrene is still evident from the infra-red spectra. The TEMphotographs of this material show thicker shell walls, presumably fromadsorbed polystyrene on the silica walls.

Example 7

Removing cores by dissolution with added ethylene diamine. 10 mg ofdried core-shell spheres are suspended in a mixture of 5 mL of ethylenediamine and 15 mL dichloromethane and stirred with a magnetic stirrerfor 1 hour. The solid is collected by centrifugation. The washingprocedure is repeated three more times followed by drying the particlesin vacuum at 60° C. for 48 hours. 3.5 mg of hollow silica spheres isobtained as white powder. Compared to Example 5 nearly all thepolystyrene core is removed by this method.

Example 8

Functionalized the hollow silica spheres with3-aminopropyl(trimethoxy)silane. 1 mg of calcined hollow silica spheres,prepared from the 100 nm templates, are suspended in 2 mL of 1%3-aminopropyl(trimethoxy)silane acetone solution, The mixture is stirredslowly for 2 hours with a magnetic stirrer followed by collecting theparticles by centrifugation. The collected particles are washed withethanol and dried in vacuum for 24 hours at room temperature.

TABLE 1 Size of hollow silica spheres isolated and its dependence on thesize of the templates and the methods of removing the polystyrene cores.Size of template (nm) 100 200 500 Diameter of core-shell 126± 210 ± 6454 ± 16 spheres (nm) Diameter of hollow 126 ± 7 205 ± 7 443 ± 21spheres after calcinations (nm) Diameter of hollow 102 ± 8 188 ± 9 397 ±15 spheres after dissolution (nm)

Example 9

Uptake of silica-NPs by dendritic cells. Immature human DCs wereincubated with FITC-labeled core-shell silica-NPs to determine cellularuptake. FITC labeled polystyrene cores were used as templated togenerate a silica shell. Immature DCs were exposed to 200 nm silica NPsfor 2 h and an image was taken in the tissue culture plate of live cells(FIG. 9 left image). All cells had taken up silica-NPs. The experimentwas repeated and 6 h after exposure to the NPs, DCs were collected andspun onto glass slides using a cyto-centrifuge. In order to determinewhether the NPs localize to the cell nucleus, the cells were fixed andthe nuclei were stained using the nuclear dye Hoechst. The slides wereanalyzed by confocal microscopy (FIG. 9, right image). A few NPs werefound to be localized in the nuclei (see white arrows) already at the 6h time point. Later time points will be investigated.

Example 10

Adsorption of plasmid DNA to hollow Silica-NPs. Hollow solvent extractedSilica -NPs were resuspended in PBS and sonicated for 15 min. Differentamounts of Silica-NPs were diluted in 700 mM NaCl and the dilution wasmixed at 10 μl per minute with equal volume of a plasmid dilution underconstant vortexing, with a total of 50 μl NP dilution and 50 μl of theDNA. After formation of the complexes, 10 μl of the mixture was resolvedon a 1% agarose gel (FIG. 6). DNA adsorption to the Silica-NPs preventsthe DNA from running into the gel. Instead, the DNA stays in the wellswith the Silica-NPs (FIG. 6 lanes 7-14), whereas free unbound plasmidruns as two bands (FIG. 6 lanes 1 and 2). A dose response of DNAadsorption to increased amounts of NPs was observed. 2 μg plasmid DNAwere completely adsorbed by 50 μl of a 0.95 mg/ml Silica-NP solution.Some free plasmid was observed when 50 μl of a 0.48 mg/ml NP solutionwere used. 6 mg of plasmid were completely adsorbed by 50 μl of a 3.8mg/ml NP solution. NPs were analyzed to determine if they could befunctionalized for enhanced DNA adsorption.

A comparison of unmodified hollow silica-NPs and modified hollowsilica-NPs with additional surface amine groups (+NH2) (FIG. 10A). Thesehollow Silica-NPs were functionalized with 3-aminopropyl (trimethoxy)silane as in Scheme II to add the additional amine groups.Amine-modified hollow silica NPs were able to almost completely adsorb 2μg of plasmid DNA when using 50 μl of a 0.48 mg/ml NP solutions (FIG.10A right gel: lane 4), whereas free plasmid DNA was detected when usingthe same amount of unmodified silica NPs (FIG. 10A left gel: lane 4).Thus, the addition of amine groups enhanced DNA adsorption. Although thesolvent extraction procedure removes 75% of the polystyrene core, toprevent toxicity in vivo, the hollow silica NPs were burned at high heat(calcination) to completely remove any residual polystyrene. The burnedhollow silica NPs with different modifications were compared for theirDNA adsorption capacity (FIG. 10B). Interestingly, burned unmodified NPsbound very little, if any DNA (FIG. 10B, left gel), whereas the solventextracted unmodified NPs effectively adsorb DNA (FIG. 10A left gel). Theburning process removes the poly-Lysine chains that remained on thesilica shell from the templating technique and thus removes the positivecharges that allow DNA adsorption. Modified silica-NPs with additionalpoly-L lysine (pLL) on the surface of burned NPs was the most potent forDNA adsorption, even 6 μg plasmid DNA was completely adsorbed by 50 μlof 0.95 mg/ml NPs (FIG. 10B, right gel). The amine modified silica-NPsonly contain short positively charged chains (see Scheme II), whereaspoly-L lysine are long chains with many amine groups. Therefore poly-Llysine coating allows for more charge-charge interactions between the NPand the DNA and therefore stronger binding capacity. The ability ofdifferent size solvent extracted unmodified hollow silica-NPs to adsorbDNA (FIG. 10C) were compared. Although all sized were able to adsorbDNA, larger 120 nm NPs showed increased DNA adsorption.

Example 11

Hollow silica-NP-DNA complexes are stable. Since the complexes weregenerated in the presence of 700 mM NaCl and the physiological saltconcentration is 150 mM, the salt was exchanged with media prior toexposure of DCs to reduce toxicity. The Silica-NP-DNA complexes weresonicated for 15 min, spun down at 6000 rpm for 10 minutes, andresuspended in culture media. To test whether the plasmid would bereleased from the Silica-NPs during these steps, aliquots were takenbefore sonication, post sonication, from the supernatant after the spin,and from the resuspended pellet (FIG. 7). At all stages of the processthe plasmid DNA remained adsorbed to the Silica-NPs and thus in thewells of the gel, indicating that neither sonication nor spinning causeda release of the plasmid DNA from the Silica-NPs. Both unmodified (U)and amine-modified (C) NPs showed the same result.

Example 12

Preparation of Silica-Iron Ethoxide Precursor. 25 mg of iron (III)ethoxide is dissolved in 1 mL of absolute ethanol. The solution is thenfiltered using a 0.22 μm syringe microfilter to remove any solids. 10 μLof the iron (III) ethoxide solution is then added to 25 μL of hydrolyzedtetramethoxysilane and vortexed for 30 seconds to give a homogenous,translucent orange solution.

Synthesis of iron doped hollow silica spheres with 200 nm aminepolystyrene beads. To 50 μL of 2.5% w/v 200 nm amine polystyrene beadswas added to 1 mL of an alcohol, either methanol or ethanol, in a 2 mLeppendorf tube. The beads were suspended and 200 μL of 0.1%poly-L-lysine solution was added and the solution was mixed for 1minute. 35 μL of the silica-iron ethoxide precursor was then added andthe solution stirred at 3000 rpm on a vortex agitator for 60 minutes atroom temperature. The yellow-orange colloid is then collected viacentrifugation. The supernatant is removed and 1 mL of deionized wateris added. The precipitate is resuspended in the water using a vortex andcollected again using centrifugation. The washing step is repeated twomore times and then the precipitate is dried in a vacuum oven at 50° C.for 24 hours. The dried precipitate is then calcined at 500° C. for 48hours to remove the polystyrene core. Up to 10% iron incorporation isobserved by EDAX in the SEM photomicrographs, which may impartbiodegradability over the long term.

Synthesis of hollow silica spheres with encapsulated magnetic iron oxidenanoparticles using 200 nm amine polystyrene beads. To 50 μL of 2.5% w/v200 nm amine polystyrene beads was added to 500 μL of a 5%dichloromethane ethanol solution (v/v) in a 2 mL eppendorf tube andsuspended using a vortex agitator. 10 μL of 1.4% (w/v) magnetic ironoxide nanoparticles (5 nm) were then added to the solution and mixed for30 seconds. The beads were allowed to swell in the solvent for 2 hours,with 10 second agitation occurring every 20 minutes. 750 μL of methanolwas then added and the solution mixed and allowed to sit at roomtemperature for 12 hours. The solvent was reduced to a volume of 250 μLand 750 μL of 0.1M phosphate buffer solution was added and the solutionmixed. 200 μL of 0.1% poly-L-lysine solution was added and the solutionwas mixed for 1 minute. 25 μL of 1M hydrolyzed tetramethoxysilane wasthen added and stirred at 3000 rpm on a vortex agitator for 10 minutes.The orange-brown precipitate is then collected in the tube using amagnet. The supernatant along with any non-magnetic particles areremoved and 1 mL of deionized water is added. The precipitate isresuspended using a vortex agitator and then recollected usingcentrifugation. This process is repeated two more times and theprecipitate is dried in a vacuum oven at 50° C. for 24 hours. The driedprecipitate is then calcined at 500° C. for 48 hours to remove thepolystyrene core. This permits magnetic manipulation of the silicananospheres.

The following references are incorporated herein in their entirety. 1.a) Pablo M. Arnal, Massimiliano Comotti, Ferdi Schuth, Angew, Chem. Int.Ed. 2006, 45, 8224-8227. b) Yufang Zhu, Jianlin Shi, Weihua Shen,Xiaoping Dong, Jingwei Feng, Meilin Ruan, Yongsheng Li, Angew, Chem.Int. Ed. 2006, 44, 5083. c) H. Wang, Z. Y. Yang, Y. F. Lu. J. Appl.Phys. 2007, 101, 033129. d) Xiangling Xu, Stanford A. Asher. J. Am.Chem. Soc. 2004, 126, 7940. e) Pu Jin, Qianwang Chen, Liqing Hao, RuifenTian, Lixin Zhang, and Lin Wang. J. Phys. Chem. B 2004, 108, 6311. f)Igor I. Slowing, Brian G. Trewyn, Supratim Giri, Victor S. -Y. Lin. Adv.Funct. Mater. 2007, 17, 1225. 2. a) S. Y. Chang, L. Liu, S. A. Asher. J.Am. Chem. Soc. 1994, 116, 6739. B) H. Yao, Y. Takada, N. Kitamura.Langmuir 1998, 14, 595. c) D. Wu, X. Ge, Z. Zhang, M. Wang, S. Zhang.Langmuir 2004, 20, 5192. d) I. Tissot, C. Novat, F. Lefebvre, E.Bourgeat-Lami. Macromolecules 2001, 34, 5737. e) K. P. Velikov, A. vanBlaaderen. Langmuir 2001, 17, 4779. 3. a) Xuefeng Ding, Kaifeng Yu,Yanqiu Jiang, Hari-Bala, Hengbin Zhang, Zichen Wang. Materials Letters2004, 58, 3618. b) W. Wu, D. Caruntu, A. Martin, M. H. Yu, C. J.O'Connor, W. L. Zhou, J-F. Chen. Journal of Magnetism and MagneticMaterials 2007, 311, 578. 4. Jeroen Cornelissen, Eric Connor, Ho-CheolKim, Victor Lee, Teddie Magibitang, Philip Rice, Willi Volksen, LindaSundberg, Robert Miller. Chem. Comm. 2003, 24, 1010. 5. Ziyi Zhong,Yadong Yin, Byron Gates, Younan Xia. Adv. Mater. 2000, 12, 206. 6. a)Frank Caruso, Heinz Lichtenfeld, Michael Giersig, Helmuth Mohward. J.Am. Chem. Soc. 1998, 120, 8623. b) Frank Caruso, Rachel A. Caruso,Helmuth Mohward. Science 1998, 282, 1111. 7. Jennifer N. Cha, Galen D.Stucky, Daniel E. Morse, Timothy J. Deming. Nature 2000, 403, 289. 8.Kjeld J. C. van Bommel, Jong Hwa Jung, Seiji Shinkai. Adv. Mater. 13,1472. 9. Bros M, et a. J Immunolo. 2003; 171(4):1825-34. 10. MordmuellerB, et al. EMBO rep. 2003; 4(1):82-7. 11. Cornelissen et al. Chem Commun.pp. 1010-1011, 2003.

Although a number of embodiments and features have been described above,it will be understood by those skilled in the art that modifications andvariations of the described embodiments and features may be made withoutdeparting from the teachings of the disclosure or the scope of theinvention as defined by the appended claims.

1. A method comprising: (a) depositing a silica-shell precursor on apolyamino acid or carboxylate functionalized template particle underneutral condition to give core-shell spheres; (b) removing the templateparticle by calcination or organic solvent to provide a hollow silicasphere.
 2. The method of claim 1, wherein the silica-shell precursor issynthesized by hydrolyzing a silicon-containing compound.
 3. The methodof claim 1, wherein the template particle further comprises a metal. 4.The method of claim 3, wherein the metal comprises an iron or magneticmetal.
 5. The method of claim 1, wherein the silica-shell precursorcomprises a silica-iron ethoxide.
 6. (canceled)
 7. The method of claim1, wherein the template particle comprises amine-functionalizedpolystyrene beads.
 8. (canceled)
 9. The method of claim 1, wherein thesize of the template particle is from about 10 nm to 1 μm.
 10. Themethod of claim 2, wherein the silicon-containing compound is selectedfrom the group consisting of tetraalkoxysilanes, trialkoxysilanes,dialkoxysilanes, tetrapropoxysilane, tetraethoxysilane,tetramethoxysilane and any combination thereof.
 11. (canceled)
 12. Themethod of claim 2, wherein the silicon-containing compound is hydrolyzedin an acidic solution.
 13. The method of claim 12, wherein the acidicsolution comprises hydrochloric acid, sulfuric acid, nitric acid or anycombination thereof. 14-16. (canceled)
 17. The method of claim 1,wherein the polyamino acids comprise monopolymers of amino acids withprimary amine groups on the backbone in solid or aqueous solution. 18.The method of claim 1, wherein the polyamino acids are about 0.1% v/waqueous solution of poly-L-lysine, poly-L-arginine and polyornithine.19-27. (canceled)
 28. A hollow sphere made according to a method ofclaim
 1. 29. A hollow sphere of claim 28, comprising a surface aminogroup or adsorbed polyamines as carriers of an oligonucleotide orpolynucleotide.
 30. A composition comprising a hollow SiO₂ nanosphereand a polynucleotide or oligonucleotide adsorbed to the nanosphere. 31.The composition of claim 30, wherein the oligonucleotide is a siRNAmolecule.
 32. The composition of claim 30, wherein the polynucleotidecomprises an expression vector.
 33. The composition of claim 32, whereinthe expression vector comprises a therapeutic or diagnostic codingsequence. 34-35. (canceled)
 36. The composition of claim 30, wherein theoligonucleotide comprises an siRNA that inhibits CMV expression.
 37. Amethod of nucleic acid delivery comprising linking an oligonucleotide orpolynucleotide to the hollow sphere of claim 28 and contacting a cell orsubject with the hollow sphere-linked nucleic acid composition. 38.(canceled)
 39. A pharmaceutical composition comprising a plurality ofhollow spheres of claim 28 or 30 in a pharmaceutically acceptablecarrier.
 40. A hollow sphere of claim 28, comprising a functional groupthat associates with a target analyte.
 41. The hollow sphere of claim28, wherein the hollow sphere is configured for quantitative detectionof the target analyte.
 42. The hollow sphere of claim 28, wherein thehollow sphere is configured for use in vivo.
 43. The hollow sphere ofclaim 40, wherein the functional group comprises a surface boundreversibly-binding receptor, the receptor specific for the targetanalyte.
 44. A method of vaccinating a subject comprising contacting thesubject with a composition comprising a hollow silica nanosphere linkedto a nucleic acid encoding a tumor antigen.
 45. The method of claim 44,wherein the composition further comprises a dendritic cell activatingagent.
 46. The method of claim 44, wherein the nanosphere furthercomprises a targeting ligand that binds to a cell surface receptor. 47.The method of claim 44 or 45, wherein nanosphere further comprises atransduction domain linked to the nanosphere.
 48. The method of claim47, wherein the transduction domain comprises a TAT peptide.